The function of the HIV-1 U5 LTR domain was examined by deleting non- overlapping one-thirds of the U5 region in the context of an infectious molecular clone of the HIV provirus. Deletion of the middle third of the HIV U5 had little effect on HIV infectivity whereas elimination of the 5' and 3' thirds rendered the resultant virus replication-incompetent by affecting encapsidation and a combination of preintegration functions (the reverse transcription and integration reactions), respectively. During the course of this work, a novel "second site" revertant was identified in which an original 26 nucleotide U5 deletion (eliminating the 3' one-third) had been extended an additional 19 nts. Characterization of the U5 deletion mutant in in vitro integration reactions revealed defective 3' processing and strand transfer activities that were partially restored in the revertant LTR substrate. A semiquantitative polymerase chain reaction procedure was used to measure the relative amounts of alternatively spliced, steady-state HIV-1 tat, rev, nef, env, and vpr mRNAs synthesized during productive virus infections. The predominant species of rev, tat, vpr, and env mRNAs contain only coding exons whereas the major (4 of 5) nef mRNAs were incompletely spliced and invariably harbored non-coding exons. This study was complemented by introducing point mutations that eliminated the use of the major splice donor or multiple splice acceptors in an infectious molecular clone of HIV-1. Mutations that inactivated certain constitutive splice sites (e.g. the major splice donor) resulted in the activation of previously unrecognized cryptic splice sites, some of which preserved biological function. Other mutations that affected "competing" splice sites (e.g. the acceptor for the first coding exon of Rev) caused alterations in the populations of viral mRNAs, and in some cases resulted in loss of infectivity. These splice site mutants provide a means for assessing the functional significance of the large redundant pool of spliced HIV RNAs and the use of alternative splicing pathways for regulating HIV expression.